Multiplex enzymatic synthesis of DNA with single-base resolution

Enzymatic DNA synthesis (EDS) is a promising benchtop and user-friendly method of nucleic acid synthesis that, instead of solvents and phosphoramidites, uses mild aqueous conditions and enzymes. For applications such as protein engineering and spatial transcriptomics that require either oligo pools or arrays with high sequence diversity, the EDS method needs to be adapted and certain steps in the synthesis process spatially decoupled. Here, we have used a synthesis cycle comprising a first step of site-specific silicon microelectromechanical system inkjet dispensing of terminal deoxynucleotidyl transferase enzyme and 3′ blocked nucleotide, and a second step of bulk slide washing to remove the 3′ blocking group. By repeating the cycle on a substrate with an immobilized DNA primer, we show that microscale spatial control of nucleic acid sequence and length is possible, which, here, are assayed by hybridization and gel electrophoresis. This work is distinctive for enzymatically synthesizing DNA in a highly parallel manner with single base control.


Fig. S2. Sequences and properties of DNA primers, synthesized probes, complementary targets and mRNA
Italics mean not synthesized. PC = photocleavable group. * Length does not include primer length. When photocleaved the product will be 23 bases longer. Underlined means can be removed from the product by cleaving enzymatically (58). ** Not including the primer. Strikethrough bases are deletion from e13 sequence. Bold font bases are substitutions to e13 sequence. Calculated by IDT's Oligoanalyzer™.

Fig. S3. DNA immobilization using Click chemistry
The graph above shows the coupling of a FAM-labelled primer (see 'labP' in S2) to a 3'azide microscope slide. Coupling is ~85% complete in 1 min. Across an entire slide the signal variation was <4%. For the composition of the coupling buffer see Materials and Methods.

Fig. S4. 1-D barcode for move-stop-print method
The figure above shows a schematic of a 1-D barcode. When printed the barcode image defines which nozzle(s) fire and how many times the nozzles fire after each move-stop. Only nozzles in 1 of the 2 nozzle rows are used (see S6). The accompanying image was captured automatically by camera under IPA control and shows the different droplet (wet) sizes generated using the barcode. The barcode was printed 5 times and the ink contained an enzyme-compatible dye (Fast Green) to improve contrast.

Fig. S5. Spot density
The table above gives the maximum number of synthesis sites for a standard 75 x 25 mm microscope slide based on the specification of the printhead. The printhead gives a print swathe 33.8 mm wide and has 800 nozzles in two interleaved rows to afford 600 dpi native resolution.
Only nozzles from the central 70% are used for printing onto the glass microscope slide. Within a row the nozzles are 1/300 th inch apart. The move-stop-print method uses only the nozzles of a single row i.e., gives 300 dpi. In this work, a print resolution of 75 dpi was chosen for synthesis. With a square lattice 75 dpi gives 16,400 sites per slide. Not all of the slide was printed on to allow the application of gaskets to hold liquids necessary for DNA hybridization and DNA cleaving.
Considering the gasket, the number of synthesis sites is closer to 10,560 spots.  Elongation ink contained 50 µM fluorescein. All slides were imaged just after printing (wet) without further treatment. The above images are the whole slide images for the GFP-TdT adsorption test with the azide and DNA slides. The order is i) after printing, ii) after automated deprotection, water washing and drying, iii) after automated proteinase K treatment, deprotection, water washing and drying, iv) after printing elongation ink.

Fig. S10. Precipitation level versus purity of nucleotide
The image above shows how precipitation of Ei-1 ink is different for two grades of dTTP-3'ONH2. Photos were taken of samples after 48 hours aging at 20 °C. Grade 1 dTTP-3'ONH2 is purer (> 95% purity) and does not contain pyrophosphates and polyphosphates. Ink formulations containing grade 1 dTTP-3'ONH2 exhibit less precipitate and no precipitate upon removing single components.

Fig. S11. Glycerol adduct (see 'Ink Formulation')
In the image above are shown the HPLC-UV chromatograms of fresh Ei-1 ink (top) and aged (24 hours at 20 °C) Ei-1 ink (middle). Also shown is the LCMS spectrum (bottom, left) of the product resulting from degradation of the 3'blocked nucleotide. It is posited to be a glycerol adduct (bottom, right). LCMS of ink Ei-2 yielded an analogous result (data not reported).